Normal somatic human cells are genetically stable and have a very low spontaneous mutation rate. In contrast, cancer cells are typically genetically unstable and accumulate thousands to hundreds of thousands of mutations in their genome. While it is known that DNA polymerase proofreading contributes to the accuracy of replication, several critical questions regarding how the loss of the proofreading function contributes to cancer remain unanswered: (1) What are the mechanisms through which proofreading dysfunction contributes to genome instability? (2) What extent do defects in polymerase proofreading play in driving cancer? and (3) To what extent do environmental factors like metal exposure influence polymerase-dependent tumor development? Here we provide evidence that cancer-associated mutations in human DNA polymerase (Pol) ?, a major replicative DNA polymerase, impair its proofreading activity, cause an increase in a unique type of mutagenesis and provide a survival advantage during metal exposure. We hypothesize that these effects have a direct influence on tumorigenesis. The main goal of this project is to test our central hypothesis that somatic mutations in Pol ? provide a selective advantage to tumor development through several, non-exclusive mechanisms, including (1) the accumulation of inactivating nonsense mutations in tumor suppressor genes; (2) resistance to oxidizing DNA damaging agents, including heavy metal exposure. This hypothesis is based on our preliminary data. Specifically, this project will 1) Establish a kinetic basis for cancer-causing Pol ? mutant alleles; 2) Define the mechanisms through which Pol ? exonuclease domain mutations (EDMs) generate their unique mutational signatures; and 3) Determine the mechanisms through which mutations in Pol ? contribute to tumor development. The proposed research is innovative due to the multidisciplinary approach that combines in vitro and in vivo studies to characterize the effects of cancer-associated Pol ? mutations on genome stability. The novel insights into how defects at the replication fork can influence genomic alterations are also innovative. This contribution is significant because it will provide new and detailed insights into the biochemical mechanisms of how replicative DNA polymerases normally prevent the acquisition of the complex diversity of mutations found in cancer genomes, as well as provide insights into the fundamental mechanisms of DNA replication. This knowledge will deepen our understanding of cancer development and can ultimately serve to inform future studies designed to modulate DNA polymerase activities toward the goal of novel cancer therapeutic strategies.